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WO2004111187A2 - Procedes d'identification de coronavirus - Google Patents

Procedes d'identification de coronavirus Download PDF

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Publication number
WO2004111187A2
WO2004111187A2 PCT/US2004/012671 US2004012671W WO2004111187A2 WO 2004111187 A2 WO2004111187 A2 WO 2004111187A2 US 2004012671 W US2004012671 W US 2004012671W WO 2004111187 A2 WO2004111187 A2 WO 2004111187A2
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WIPO (PCT)
Prior art keywords
coronavirus
sequence
virus
bioagent
primers
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WO2004111187A3 (fr
Inventor
David J. Ecker
Steven A. Hofstadler
Rangarajan Sampath
Lawrence B. Blyn
Thomas A. Hall
Christian Massire
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Ionis Pharmaceuticals Inc
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Isis Pharmaceuticals Inc
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Priority to CA002521508A priority Critical patent/CA2521508A1/fr
Priority to AU2004248107A priority patent/AU2004248107A1/en
Priority to EP04775904A priority patent/EP1623013A4/fr
Priority to JP2006532460A priority patent/JP2007523629A/ja
Publication of WO2004111187A2 publication Critical patent/WO2004111187A2/fr
Anticipated expiration legal-status Critical
Publication of WO2004111187A3 publication Critical patent/WO2004111187A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

  • the present invention relates generally to the field of genetic identification and quantitation of coronaviruses and provides methods, compositions and kits useful for this purpose when combined with molecular mass analysis.
  • Coronaviruses a genus in the family Coronoviridae, are large, enveloped RNA viruses that cause highly prevalent diseases in humans and domestic animals. Coronavirus particles are irregularly-shaped, 60-220 run in diameter, with an outer envelope bearing distinctive, "club- shaped” peplomers. This "crown-like" appearance gives the family its name. Coronaviruses have the largest genomes of all RNA viruses and replicate by a unique mechanism which results in a high frequency of recombination. Virions mature by budding at intracellular membranes and infection with some coronaviruses induces cell fusion.
  • HcoVs human coronaviruses
  • the 5' 20kb of the (+)sense genome is translated to produce a viral polymerase, which is believed to produce a full-length (-)sense strand which, in turn, is used as a template to produce mRNA as a "nested set" of transcripts, all with an identical 5' non-translated leader sequence of 72 nucleotides and coincident 3' polyadenylated ends.
  • Each mRNA is monocistronic, the genes at the 5' end being translated from the longest mRNA.
  • These unusual cytoplasmic structures are produced not by splicing (post-transcriptional modification) but by the polymerase during transcription. Coronaviruses infect a variety of mammals and birds. The exact number of human isolates is not known as many cannot be grown in culture. In humans, they cause: respiratory infections (common), including Severe Acute Respiratory Syndrome (SARS), and enteric infections.
  • SARS Severe Acute Respiratory Syndrome
  • Coronaviruses are transmitted by aerosols of respiratory secretions, by the fecal-oral route, and by mechanical transmission. Most virus growth occurs in epithelial cells. Occasionally the liver, kidneys, heart or eyes may be infected, as well as other cell types such as macrophages. In cold-type respiratory infections, growth appears to be localized to the epithelium of the upper respiratory tract, but there is currently no adequate animal model for the human respiratory coronaviruses. Clinically, most infections cause a mild, self-limited disease (classical "cold" or upset stomach), but there may be rare neurological complications. Coronavirus infection is very common and occurs worldwide. The incidence of infection is strongly seasonal, with the greatest incidence in children in winter. Adult infections are less common.
  • coronavirus serotypes The number of coronavirus serotypes and the extent of antigenic variation are unknown. Re-infections appear to occur throughout life, implying multiple serotypes (at least four are known) and/or antigenic variation, hence the prospects for immunization appear bleak.
  • SARS severe Acute Respiratory Syndrome
  • Typical laboratory findings include lymphopenia (reduced lymphocyte numbers) and mildly elevated aminotransferase levels (indicating liver damage). Death may result from progressive respiratory failure due to alveolar damage.
  • SARS virus can be grown in Vero cells (a primate fibroblast cell line) - a novel property for HCoVs, most of which cannot be cultivated. In these cells, virus infection results in a cytopathic effect, and budding of coronavirus-like particles from the endoplasmic reticulum within infected cells.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the second test an immunofluorescence assay (IFA) detects antibodies reliably as of day 10 of infection. It shares the defect of the ELISA test in that test subjects have become infective prior to IFA-based diagnosis. Moreover, the IFA test is a demanding and comparatively slow test that requires the growth of virus in cell culture.
  • the third test is a polymerase chain reaction (PCR) molecular test for detection of SARS virus genetic material is useful in the early stages of infection but undesirably produces false- negatives.
  • PCR polymerase chain reaction
  • PCR test may fail to detect persons who actually carry the virus, even in conjunction with clinical diagnostic evaluation, creating a dangerous sense of false security in the face of a potential epidemic of a virus that is known to spread easily in close person-to- 5 person contact (WHO. Severe acute respiratory syndrome (SARS). WkIy Epidemiol. Rec. 2003, 78, 121-122).
  • nucleic acid tests for infectious diseases are largely based upon amplifications using primers and probes designed to detect specific bioagents. Because prior knowledge of nucleic acid sequence information is required to develop these tests they are not able to identify
  • infectious bioagents still relies largely on culture and microscopy, which were as important in the recent identification of the SARS coronavirus as they were in the discovery of the human immunodeficiency virus two decades ago.
  • coronaviruses (Stephensen, C. B., Casebolt, D. B. Gangopadhyay, N. N. Vir. Res. 60, 181-189 (1999)), enteroviruses (M. S. Oberste, K. Maher, M. A. Pallansch, J Virol. 76, 1244-51 (2002); M. S. Oberste, W. A. Nix, K. Maher, M. A. Pallansch, J. Clin. Virol. 26, 375-7 (2003); M. S. Oberste, W. A. Nix, D. R. Kilpatrick, M. R. Flemister, M. A. Pallansch, Virus Res. 91, 241-8(2003)), retroid viruses(D. H. Mack, J. J. Sninsky, Proc. Natl. Acad. Sci. U. S. A. 85, 6977-
  • Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated.
  • high- resolution MS alone fails to perform against unknown or bioengineered agents, or in environments where there is a high background level of bioagents ("cluttered" background).
  • Low-resolution MS can fail to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample.
  • DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism.
  • Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments.
  • Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 10:377-382, 1996).
  • MALDI-TOF matrix-assisted laser desorption ionization- time of flight
  • U.S. Patent No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences which comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism.
  • U.S. Patent No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods.
  • WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry.
  • WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.
  • PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS.
  • U.S. Patent No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.
  • WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry.
  • Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation.
  • Methods of sequencing a nucleic acid via mass spectrometry methods are also described.
  • WO 97/37041, WO 99/31278 and US Patent No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry.
  • US Patent Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.
  • the present invention provides a novel approach for rapid, sensitive, and high- throughput identification of coronaviruses and includes the capability of identification of coronaviruses not yet observed and characterized.
  • the methods described can be applied to additional viral families to cover a broad range of potential newly emerging viruses, or to bacterial, protozoal or fungal pathogens for epidemic disease surveillance in the future.
  • the present invention is directed to, inter alia, methods of identification of one or more unknown coronaviruses in a sample by obtaining coronavirus RNA from the sample, obtaining corresponding DNA from the RNA, amplifying the DNA with one or more pairs of oligonucleotide primers that bind to conserved regions of a coronavirus genome which are flanked a variable region of the coronavirus genome, determining the molecular masses or base compositions of the one or more amplification products and comparing the molecular masses or base compositions with calculated or experimentally determined molecular masses or base compositions, wherein one or more matches identifies the unknown coronavirus.
  • the present invention is also directed to methods of tracking the spread of a specific coronavirus comprising: obtaining a plurality of samples containing a specific coronavirus from a plurality of different locations, identifying the specific coronavirus in a subset of the plurality of samples using the method described in the paragraph above, wherein the corresponding locations of the members of the subset indicate the spread of the specific coronavirus to the corresponding locations.
  • the present invention is also directed to pairs of primers wherein each member of each pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following intelligent primer pair sequences: SEQ ID NOs: 5:6, 7:8, 9:8, 9:10, 11:8, 11 :10 or 9:10.
  • the present invention is also directed to individual primers within each of the primer pairs described herein.
  • the present invention is also directed to bioagent identifying amplicons for identification of a coronavirus comprising an isolated polynucleotide of about 45 to about 150 nucleobases in length produced by the process of amplification of nucleic acid from a coronavirus genome with a primer pair wherein each primer of the pair is of a length of about 12 to about 35 nucleobases and the bioagent identifying amplicon provides identifying information about the coronavirus.
  • the present invention is also directed to methods for simultaneous determination of the identity and quantity of an unknown coronavirus in a sample comprising: contacting a sample with a pair of primers and a known quantity of a calibration polynucleotide comprising a calibration sequence, simultaneously amplifying nucleic acid from the unknown coronavirus with the pair of primers and amplifying nucleic acid from the calibration polynucleotide in the sample with the pair of primers to obtain a first amplification product comprising a bioagent identifying amplicon and a second amplification product comprising a calibration amplicon, subjecting the sample to molecular mass analysis wherein the result of the mass analysis comprises molecular mass and abundance data for the bioagent identifying amplicon and the calibration amplicon, and distinguishing the bioagent identifying amplicon from the calibration amplicon based on molecular mass wherein the molecular mass of the bioagent identifying amplicon identifies the coronavirus and comparison of bioagent identifying ampli
  • the present invention is also directed to isolated polynucleotides for determining the quantity of a bioagent in a sample comprising SEQ ID NOs: 102, and 103 as well as vectors comprising of SEQ ID NOs: 102, 103 and 104.
  • kits comprising one or more pairs of primers, or individual primers, wherein each member of each pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following intelligent primer pair sequences: SEQ ID NOs: 5:6, 7:8, 9:8, 9:10, 11:8, 11:10 or 9:10.
  • Figures IA- IH and Figure 2 are consensus diagrams that show examples of conserved regions of 16S rRNA (Fig. IA, 1A-2, 1A-3, 1A-4 and 1A-5), 23S rRNA (3'-half, Fig. IB-I, IB-
  • DNA segments encoding these regions are suitable for use as templates for generation of bioagent identifying amplicons.
  • Lines with arrows are examples of regions (in corresponding DNA) to which intelligent primer pairs for PCR are designed.
  • the label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram. Bases in capital letters are greater than 95% conserved; bases in lower case letters are 90-95% conserved, filled circles are 80-90% conserved; and open circles are less than 80% conserved.
  • the label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
  • the nucleotide sequence of the 16S rRNA consensus sequence is SEQ ID NO:3 and the nucleotide sequence of the 23S rRNA consensus sequence is SEQ ID NO:4.
  • Figure 2 shows a typical primer amplified region from the 16S rRNA Domain III shown in Figure IA-I.
  • Figure 3 is a schematic diagram showing conserved regions in RNase P. Bases in capital letters are greater than 90% conserved; bases in lower case letters are 80-90% conserved; filled circles designate bases which are 70-80% conserved; and open circles designate bases that are less than 70% conserved.
  • Figure 4 is a schematic diagram of base composition signature determination using nucleotide analog "tags" to determine base composition signatures.
  • Figure 5 shows the deconvoluted mass spectra of a Bacillus anthracis region with and without the mass tag phosphorothioate A (A*).
  • the two spectra differ in that the measured molecular weight of the mass tag-containing sequence is greater than the unmodified sequence.
  • Figure 6 is a process diagram illustrating the primer selection process.
  • candidate target sequences are identified (200) from which nucleotide alignments are created (210) and analyzed (220).
  • Primers are then designed by selecting appropriate priming regions (230) which then makes possible the selection of candidate primer pairs (240).
  • the primer pairs are then subjected to in silico analysis by electronic PCR (ePCR) (300) wherein bioagent identifying amplicons are obtained from sequence databases such as GenBank or other sequence collections (310) and checked for specificity in silico (320).
  • ePCR electronic PCR
  • Bioagent identifying amplicons obtained from GenBank sequences (310) can also be analyzed by a probability model which predicts the capability of a given amplicon to identify unknown bioagents such that the base compositions of amplicons with favorable probability scores are then stored in a base composition database (325).
  • base compositions of the bioagent identifying amplicons obtained from the primers and GenBank sequences can be directly entered into the base composition database (330).
  • Candidate primer pairs (240) are validated by in vitro amplification by a method such as PCR analysis (400) of nucleic acid from a collection of organisms (410). Amplification products thus obtained are analyzed to confirm the sensitivity, specificity and reproducibility of the primers used to obtain the amplification products (420).
  • Figure 7 indicates two coronavirus genomic regions used to generate bioagent identifying amplicons for coronaviruses.
  • the two primer pair regions are mapped onto the SARS CoV (TOR2, NC_004718.3: SEQ ID NO: 85) genome coordinates.
  • the RdRp amplicon corresponds to positions 15,132-15,218 and the nspl l amplicon corresponds to positions 19,098- 19,234 of the same sequence. Multiple sequence alignments of additional available coronavirus sequences corresponding to these two amplified regions are shown.
  • Coronavirus isolates that were sequenced as part of this work are shown with an asterisk. All other sequences were obtained from GenBank.
  • nspl 1 sequences for PHEV and TCoV are not available and are shown with a dashed line. Forward and reverse primer regions are highlighted. 5' ends of all primers were designed with a thymidine (T) nucleotide which acts to minimize the addition of non- templated A residues during PCR. For each specific coronavirus, the positions that are mismatched compared to the primer sequence are shown. The complement of the actual primer is shown for the reverse primers. The region between the forward and reverse primers for each virus varies among different coronaviruses (not shown) and provides the signature for resolving them by molecular mass or base composition. Primer positions chemically modified with propyne groups are highlighted. The structures of 5-propynyldeoxycytidine and 5- propynyldeoxyurididine nucleotides used in primers are shown.
  • Figure 8 is an ESI-FTICR mass spectrum measurement of the PCR amplicon from the
  • SARS coronavirus obtained using the propynylated RdRp primer pairs.
  • the electrospray ionization conditions separate the sense and antisense strands of the PCR products (500). Multiple charge states are observed across the m/z range shown (510) from which is obtained an expanded view of the isotope envelope of the (M-27H+)27- species (520).
  • the derived molecular masses (530) for the sense amplicon strand is 27298.518 from which is calculated
  • This base composition corresponds to the base composition calculated based on the bioagent identifying amplicon of the genomic sequence of the SARS coronavirus.
  • Figure 9 shows a series of mass spectra which was used to identify three human coronaviruses in a mixture.
  • the deconvoluted (neutral mass) mass spectra obtained for the RdRp primer for the three human coronaviruses, HCoV-229E, HCoV-OC43 and SARS CoV, that were tested individually and in a mixture are shown. Forward and reverse amplicons are shown with the measured monoisotopic masses for each strand.
  • Figure 10 indicates three dimensional plots of experimentally determined base compositions (solid cones) and calculated base compositions (spheres) for bioagent identifying amplicons of coronaviruses obtained with a RdRp primer set and a nspl l primer set.
  • the position of the base composition indicates the intersection of A, C and T content in each base composition, with G content illustrated in a "pseudo-fourth" dimension indicated by the angle of rotation of the cone.
  • the experimentally-determined base compositions were in agreement with the calculated base compositions except for the canine coronavirus isolate (CcoV) which was found to have a composition that differed from that calculated based on available sequence information by a single T to C substitution (indicated by a dashed line).
  • CcoV canine coronavirus isolate
  • Figure 11 shows a mass spectrum containing mass peaks corresponding to the PCR internal standard calibrant amplicon at a dilution corresponding to a nucleic acid copy number of 3 x 10 4 and a SARS coronavirus bioagent identifying amplicon.
  • the present invention provides methods for detection and identification of bioagents in an unbiased manner using "bioagent identifying amplicons.”
  • "Intelligent primers” are selected to hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions to yield a bioagent identifying amplicon which can be amplified and which is amenable to molecular mass determination.
  • the molecular mass then provides a means to uniquely identify the bioagent without a requirement for prior knowledge of the possible identity of the bioagent.
  • the molecular mass or corresponding "base composition signature" (BCS) of the amplification product is then matched against a database of molecular masses or base composition signatures.
  • BCS base composition signature
  • the method can be applied to rapid parallel "multiplex" analyses, the results of which can be employed in a triangulation identification strategy.
  • the present method provides rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for bioagent detection and identification.
  • a “bioagent” is any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus.
  • bioagents include, but are not limited, to cells, including but not limited to human clinical samples, bacterial cells and other pathogens), viruses, fungi, protists, parasites, and pathogenicity markers (including but not limited to: pathogenicity islands, antibiotic resistance genes, virulence factors, toxin genes and other bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered.
  • a "pathogen” is a bioagent which causes a disease or disorder.
  • RNAse P Figure 3
  • SRP signal recognition particle
  • genes encode proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like.
  • proteins are DNA polymerase III beta, elongation factor TU, heat shock protein groEL, RNA polymerase beta, phosphoglycerate kinase, NADH dehydrogenase, DNA ligase, DNA topoisomerase and elongation factor G.
  • Operons can also be targeted using the present method.
  • One example of an operon is the bfp operon from enteropathogenic E. coli.
  • Multiple core chromosomal genes can be used to classify bacteria at a genus or genus species level to determine if an organism has threat potential.
  • the methods can also be used to detect pathogenicity markers (plasmid or chromosomal) and antibiotic resistance genes to confirm the threat potential of an organism and to direct countermeasures.
  • At least one polynucleotide segment is amplified to facilitate detection and analysis in the process of identifying the bioagent.
  • nucleic acid segments which provide enough variability to distinguish each individual bioagent and whose molecular masses are amenable to molecular mass determination are herein described as "bioagent identifying amplicons.”
  • amplicon refers to a segment of a polynucleotide which is amplified in an amplification reaction.
  • bioagent identifying amplicons are from about 45 nucleobases to about 150 nucleobases in length.
  • Pre-bioagent identifying amplicons are amplicons which may greatly exceed about 45 to about 150 nucleobases in length and which contain sites for cleavage (by restriction endonucleases, for example) to yield bioagent identifying amplicons which are fragments of a given pre-bioagent identifying amplicon and which are amenable to molecular mass analysis.
  • "intelligent primers” are primers that are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which ideally provide enough variability to distinguish each individual bioagent, and which are amenable to molecular mass analysis.
  • bioagent identifying amplicons are ideally specific to the identity of the bioagent.
  • a plurality of bioagent identifying amplicons selected in parallel for distinct bioagents which contain the same conserved sequences for hybridization of the same pair of intelligent primers are herein defined as "correlative bioagent identifying amplicons.”
  • the bioagent identifying amplicon is a portion of a ribosomal RNA (rRNA) gene sequence.
  • rRNA ribosomal RNA
  • rRNA genes Like many genes involved in core life functions, rRNA genes contain sequences that are extraordinarily conserved across bacterial domains interspersed with regions of high variability that are more specific to each species. The variable regions can be utilized to build a database of base composition signatures. The strategy involves creating a structure-based alignment of sequences of the small (16S) and the large (23S) subunits of the rRNA genes.
  • regions that are useful as bioagent identifying amplicons include: a) between about 80 and 100%, or greater than about 95% identity among species of the particular bioagent of interest, of upstream and downstream nucleotide sequences which serve as sequence amplification primer sites; b) an intervening variable region which exhibits no greater than about 5% identity among species; and c) a separation of between about 30 and 1000 nucleotides, or no more than about 50-250 nucleotides, or no more than about 60-100 nucleotides, between the conserved regions.
  • the conserved sequence regions of the chosen bioagent identifying amplicon must be highly conserved among all Bacillus species while the variable region of the bioagent identifying amplicon is sufficiently variable such that the molecular masses of the amplification products of all species of Bacillus are distinguishable.
  • Bioagent identifying amplicons amenable to molecular mass determination are either of a length, size or mass compatible with the particular mode of molecular mass determination or compatible with a means of providing a predictable fragmentation pattern in order to obtain predictable fragments of a length compatible with the particular mode of molecular mass determination.
  • Such means of providing a predictable fragmentation pattern of an amplification product include, but are not limited to, cleavage with restriction enzymes or cleavage primers, for example.
  • Identification of bioagents can be accomplished at different levels using intelligent primers suited to resolution of each individual level of identification. "Broad range survey" intelligent primers are designed with the objective of identifying a bioagent as a member of a particular division of bioagents.
  • a “bioagent division” is defined as group of bioagents above the 5 species level and includes but is not limited to: orders, families, classes, clades, genera or other such groupings of bioagents above the species level.
  • members of the Bacillus/Clostridia group or gamma-proteobacteria group may be identified as such by employing broad range survey intelligent primers such as primers which target 16S or 23 S ribosomal RNA.
  • broad range survey intelligent primers are capable of identification of bioagents at the species level.
  • One main advantage of the detection methods of the present invention is that the broad range survey intelligent primers need not be specific for a particular bacterial species, or even genus, such as Bacillus or Streptomyces. Instead, the primers recognize highly conserved regions across hundreds of bacterial species including, but not
  • the same broad range survey intelligent primer pair can be used to identify any desired bacterium because it will bind to the conserved regions that flank a variable region specific to a single species, or common to several bacterial species, allowing unbiased nucleic acid amplification of the intervening sequence and determination of its molecular weight and base composition.
  • primers used in the present method bind to one or more of these regions or portions thereof.
  • flanking rRNA primer sequences serve as good intelligent primer binding sites to amplify the nucleic acid region of interest for most, if not all,
  • the intervening region between the sets of primers varies in length and/or composition, and thus provides a unique base composition signature.
  • Examples of intelligent primers that amplify regions of the 16S and 23S rRNA are shown in Figures 1A-1H.
  • a typical primer amplified region in 16S rRNA is shown in Figure 2.
  • the arrows represent primers that bind to highly conserved regions of the DNA encoding these regions, which flank a variable
  • the amplified region corresponds to the stem-loop structure under "1100-1188.” It is advantageous to design the broad range survey intelligent primers to minimize the number of primers required for the analysis, and to allow detection of multiple members of a bioagent division using a single pair of primers.
  • the advantage of using broad range survey intelligent primers is that once a bioagent is broadly identified, the process of further identification at species and sub-species levels is facilitated by directing the choice of additional intelligent primers.
  • "Division-wide" intelligent primers are designed with an objective of identifying a bioagent at the species level.
  • a Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis can be distinguished from each other using division-wide intelligent primers.
  • Division- wide intelligent primers are not always required for identification at the species level because broad range survey intelligent primers may provide sufficient identification resolution to accomplishing this identification objective.
  • Drill-down intelligent primers are designed with an objective of identifying a subspecies characteristic of a bioagent.
  • a “sub-species characteristic” is defined as a property imparted to a bioagent at the sub-species level of identification as a result of the presence or absence of a particular segment of nucleic acid.
  • Such sub-species characteristics include, but are not limited to, strains, sub-types, pathogenicity markers such as antibiotic resistance genes, pathogenicity islands, toxin genes and virulence factors. Identification of such sub-species characteristics is often critical for determining proper clinical treatment of pathogen infections.
  • a representative process flow diagram used for primer selection and validation process is outlined in Figure 6. Many of the important pathogens, including the organisms of greatest concern as biological weapons agents, have been completely sequenced. This effort has greatly facilitated the design of primers and probes for the detection of bacteria. Partial or full-length sequences from over 225 bacterial genomes have been obtained and sequence alignments have been generated for essential genes that are conserved either broadly across all organisms or within members of specific, related phylogenetic groups. In bacteria, for instance, alignments have been generated from over 170 housekeeping genes that are present in almost all major bacterial divisions. These genes have been used for identification of broad diagnostic primers. PCR primer selection and optimization has been largely automated.
  • intelligent primer hybridization sites are highly conserved in order to facilitate the hybridization of the primer.
  • intelligent primers can be chemically modified to improve the efficiency of hybridization.
  • intelligent primers may contain one or more universal bases. Because any variation (due to codon wobble in the 3 rd position) in the conserved regions among species is likely to occur in the third position of a DNA triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a "universal nucleobase.” For example, under this "wobble” pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C.
  • inosine (I) binds to U, C or A
  • guanine (G) binds to U or C
  • uridine (U) binds to U or C.
  • nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et ah, Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK (Hill et ah), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et ah, Nucleosides and Nucleotides, 1995, 14, 1053-1056) or the purine analog l-(2-deoxy- ⁇ -D-ribofuranosyl)-imidazole-4- carboxamide (SaIa et ah, Nuch Acids Res., 1996, 24, 3302-3306).
  • nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et ah, Nucleosides and Nucleotides, 1995, 14, 1001-1003)
  • the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide.
  • nucleotide analogs include, but are not limited to, 2,6-diaminopurine which binds to thymine, 5-propynyluracil which binds to adenine and 5- propynylcytosine and phenoxazines, including G-clamp, which binds to G.
  • Propynylated pyrimidines are described in U.S. Patent Nos.
  • non-template primer tags are used to increase the melting temperature (T m ) of a primer-template duplex in order to improve amplification efficiency.
  • a non-template tag is designed to hybridize to at least three consecutive A or T nucleotide residues on a primer which are complementary to the template.
  • A can be replaced by C or G and T can also be replaced by C or G.
  • the extra hydrogen bond in a G-C pair relative to a A-T pair confers increased stability of the primer-template duplex and improves amplification efficiency.
  • propynylated tags may be used in a manner similar to that of the non-template tag, wherein two or more 5-propynylcytidine or 5-propynyluridine residues replace template matching residues on a primer.
  • a primer contains a modified internucleoside linkage such as a phosphorothioate linkage, for example.
  • a theoretically ideal bioagent detector would identify, quantify, and report the complete nucleic acid sequence of every bioagent that reached the sensor.
  • the complete sequence of the nucleic acid component of a pathogen would provide all relevant information about the threat, including its identity and the presence of drug-resistance or pathogenicity markers. This ideal has not yet been achieved.
  • the present invention provides a straightforward strategy for obtaining information with the same practical value based on analysis of bioagent identifying amplicons by molecular mass determination.
  • a molecular mass of a given bioagent identifying amplicon alone does not provide enough resolution to unambiguously identify a given bioagent.
  • the molecular mass of the bioagent identifying amplicon obtained using the intelligent primer pair "16S_971" would be 55622 Da for both E. coli and Salmonella typhimurium.
  • additional intelligent primers are employed to analyze additional bioagent identifying amplicons, a "triangulation identification” process is enabled.
  • the "16S_1100" intelligent primer pair yields molecular masses of 55009 and 55005 Da for E. coli and Salmonella typhimurium, respectively.
  • the "23S_855" intelligent primer pair yields molecular masses of 42656 and 42698 Da for E. coli and Salmonella typhimurium, respectively.
  • the second and third intelligent primer pairs provided the additional "fingerprinting" capability or resolution to distinguish between the two bioagents.
  • the triangulation identification process is pursued by measuring signals from a plurality of bioagent identifying amplicons selected within multiple core genes. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. In this process, after identification of multiple core genes, alignments are created from nucleic acid sequence databases.
  • bioagent identifying amplicons are selected to distinguish bioagents based on specific genomic differences. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol, 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.
  • the triangulation identification process can be pursued by characterization of bioagent identifying amplicons in a massively parallel fashion using the polymerase chain reaction (PCR), such as multiplex PCR, and mass spectrometric (MS) methods. Sufficient quantities of nucleic acids should be present for detection of bioagents by MS.
  • PCR polymerase chain reaction
  • MS mass spectrometric
  • PCR requires one or more pairs of oligonucleotide primers that bind to regions which flank the target sequence(s) to be amplified. These primers prime synthesis of a different strand of DNA 5 with synthesis occurring in the direction of one primer towards the other primer.
  • the primers, DNA to be amplified, a thermostable DNA polymerase (e.g. Tag polymerase), the four deoxynucleotide triphosphates, and a buffer are combined to initiate DNA synthesis.
  • the solution is denatured by heating, then cooled to allow annealing of newly added primer, followed by another round of DNA synthesis. This process is typically repeated for about 30 cycles, resulting in amplification of the target sequence.
  • PCR ligase chain reaction
  • SDA strand displacement amplification
  • the detection scheme for the PCR products generated from the bioagent(s) incorporates at least three features. First, the technique simultaneously detects and differentiates multiple (generally about 6-10) PCR products. Second, the technique provides a molecular mass that uniquely identifies the bioagent from the possible primer sites. Finally, the detection technique is rapid, allowing multiple PCR reactions to be run in parallel.
  • Mass spectrometry (MS)-based detection of PCR products provides a means for determination of BCS which has several advantages.
  • MS is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product is identified by its molecular mass.
  • the current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample.
  • An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons.
  • Intact molecular ions can be generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB).
  • ES electrospray ionization
  • MALDI matrix-assisted laser desorption ionization
  • FAB fast atom bombardment
  • MALDI of nucleic acids along with examples of matrices for use in MALDI of nucleic acids, are described in WO 98/54751.
  • large DNAs and RNAs, or large amplification products therefrom can be digested with restriction endonucleases prior to ionization.
  • an amplification product that was 10 kDa could be digested with a series of restriction endonucleases to produce a panel of, for example, 100 Da fragments. Restriction endonucleases and their sites of action are well known to the skilled artisan. In this manner, mass spectrometry can be performed for the purposes of restriction mapping.
  • Electrospray ionization mass spectrometry is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.
  • the mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, and triple quadrupole.
  • FT-ICR-MS Fourier transform ion cyclotron resonance mass spectrometry
  • ion trap ion trap
  • quadrupole quadrupole
  • magnetic sector magnetic sector
  • TOF time of flight
  • Q-TOF Q-TOF
  • triple quadrupole triple quadrupole
  • the mass spectrometric techniques which can be used in the present invention include, but are not limited to, tandem mass spectrometry, infrared multiphoton dissociation and pyrolytic gas chromatography mass spectrometry (PGC-MS).
  • the bioagent detection system operates continually in bioagent detection mode using pyrolytic GC-MS without PCR for rapid detection of increases in biomass (for example, increases in fecal contamination of drinking water or of germ warfare agents).
  • a continuous sample stream flows directly into the PGC-MS combustion chamber.
  • a PCR process is automatically initiated.
  • Bioagent presence produces elevated levels of large molecular fragments from, for example, about 100-7,000 Da which are observed in the PGC-MS spectrum.
  • the observed mass spectrum is compared to a threshold level and when levels of biomass are determined to exceed a predetermined threshold, the bioagent classification process described hereinabove (combining PCR and MS, such as FT-ICR MS) is initiated.
  • alarms or other processes are also initiated by this detected biomass level.
  • the accurate measurement of molecular mass for large DNAs is limited by the adduction of cations from the PCR reaction to each strand, resolution of the isotopic peaks from natural abundance 13 C and 15 N isotopes, and assignment of the charge state for any ion.
  • the cations are removed by in-line dialysis using a flow-through chip that brings the solution containing the PCR products into contact with a solution containing ammonium acetate in the presence of an electric field gradient orthogonal to the flow.
  • the latter two problems are addressed by operating with a resolving power of > 100,000 and by incorporating isotopically depleted nucleotide triphosphates into the DNA.
  • the resolving power of the instrument is also a consideration.
  • Tandem MS involves the coupled use of two or more stages of mass analysis where both the separation and detection steps are based on mass spectrometry.
  • the first stage is used to select an ion or component of a sample from which further structural information is to be obtained.
  • the selected ion is then fragmented using, e.g., blackbody irradiation, infrared multiphoton dissociation, or collisional activation.
  • ESI electrospray ionization
  • This activation leads to dissociation of glycosidic bonds and the phosphate backbone, producing two series of fragment ions, called the w-series (having an intact 3' terminus and a 5' phosphate following internal cleavage) and the ⁇ -Base series(having an intact 5' terminus and a 3' furan).
  • the second stage of mass analysis is then used to detect and measure the mass of these resulting fragments of product ions.
  • Such ion selection followed by fragmentation routines can be performed multiple times so as to essentially completely dissect the molecular sequence of a sample.
  • a nucleotide analog or "tag” is incorporated during amplification (e.g., a 5- (trifluoromethyl) deoxythymidine triphosphate) which has a different molecular weight than the unmodified base so as to improve distinction of masses.
  • tags are described in, for example, PCT WO97/33000, which is incorporated herein by reference in its entirety. This further limits the number of possible base compositions consistent with any mass.
  • 5- (trifluoromethyl)deoxythymidine triphosphate can be used in place of dTTP in a separate nucleic acid amplification reaction.
  • Measurement of the mass shift between a conventional amplification product and the tagged product is used to quantitate the number of thymidine nucleotides in each of the single strands. Because the strands are complementary, the number of adenosine nucleotides in each strand is also determined.
  • the number of G and C residues in each strand is determined using, for example, the cytosine analog 5-methylcytosine (5-meC) or 5- propynylcytosine.
  • the combination of the A/T reaction and G/C reaction, followed by molecular weight determination, provides a unique base composition. This method is summarized in Figure 4 and Table 1.
  • the mass tag phosphorothioate A (A*) was used to distinguish a Bacillus anthracis cluster.
  • the B. anthracis (Ai 4 G 9 C 14 T 9 ) had an average MW of 14072.26, and the B. anthracis (A 1 A* UGgC 14 T 9 ) had an average molecular weight of 14281.11 and the phosphorothioate A had an average molecular weight of +16.06 as determined by ESI-TOF MS.
  • the deconvoluted spectra are shown in Figure 5. In another example, assume the measured molecular masses of each strand are
  • the measured number of dT and dA residues are (30,28) and (28,30). If the molecular mass is accurate to 100 ppm, there are 7 possible combinations of dG+dC possible for each strand. However, if the measured molecular mass is accurate to 10 ppm, there are only 2 combinations of dG+dC, and at 1 ppm accuracy there is only one possible base composition for each strand.
  • Signals from the mass spectrometer may be input to a maximum-likelihood detection and classification algorithm such as is widely used in radar signal processing.
  • the detection processing uses matched filtering of BCS observed in mass-basecount space and allows for detection and subtraction of signatures from known, harmless organisms, and for detection of unknown bioagent threats. Comparison of newly observed bioagents to known bioagents is also possible, for estimation of threat level, by comparing their BCS to those of known organisms and to known forms of pathogenicity enhancement, such as insertion of antibiotic resistance genes or toxin genes.
  • Processing may end with a Bayesian classifier using log likelihood ratios developed from the observed signals and average background levels.
  • the program emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants.
  • Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents.
  • a genomic sequence database e.g. GenBank
  • GenBank is used to define the mass basecount matched filters.
  • the database contains known threat agents and benign background organisms. The latter is used to estimate and subtract the signature produced by the background organisms.
  • a maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance.
  • a base composition signature is the exact base composition determined from the molecular mass of a bioagent identifying amplicon.
  • a BCS provides an index of a specific gene in a specific organism.
  • Base compositions like sequences, vary slightly from isolate to isolate within species. It is possible to manage this diversity by building "base composition probability clouds” around the composition constraints for each species. This permits identification of organisms in a fashion similar to sequence analysis. A "pseudo four-dimensional plot" can be used to visualize the concept of base composition probability clouds.
  • Optimal primer design requires optimal choice of bioagent identifying amplicons and maximizes the separation between the base composition signatures of individual bioagents. Areas where clouds overlap indicate regions that may result in a misclassification, a problem which is overcome by selecting primers that provide information from different bioagent identifying amplicons, ideally maximizing the separation of base compositions.
  • one aspect of the utility of an analysis of base composition probability clouds is that it provides a means for screening primer sets in order to avoid potential misclassifications of BCS and bioagent identity.
  • base composition probability clouds provide a means for predicting the identity of a bioagent whose exact measured BCS was not previously observed and/or indexed in a BCS database due to evolutionary transitions in its nucleic acid sequence. It is important to note that, in contrast to probe-based techniques, mass spectrometry determination of base composition does not require prior knowledge of the composition in order to make the measurement, only to interpret the results.
  • the present invention provides bioagent classifying information similar to DNA sequencing and phylogenetic analysis at a level sufficient to detect and identify a given bioagent.
  • Another embodiment of the present invention is a method of surveying bioagent samples that enables detection and identification of all bacteria for which sequence information is available using a set of twelve broad-range intelligent PCR primers. Six of the twelve primers are "broad range survey primers" herein defined as primers targeted to broad divisions of bacteria (for example, the Bacillus/Clostridia group or gamma-proteobacteria).
  • the other six primers of the group of twelve primers are "division-wide" primers herein defined as primers which provide more focused coverage and higher resolution.
  • This method enables identification of nearly 100% of known bacteria at the species level.
  • a further example of this embodiment of the present invention is a method herein designated “survey/drill-down" wherein a subspecies characteristic for detected bioagents is obtained using additional primers. Examples of such a subspecies characteristic include but are not limited to: antibiotic resistance, pathogenicity island, virulence factor, strain type, sub-species type, and clade group.
  • bioagent detection, confirmation and a subspecies characteristic can be provided within hours.
  • the survey/drill-down method can be focused to identify bioengineering events such as the insertion of a toxin gene into a bacterial species that does not normally make the toxin.
  • Coronaviruses represent RNA virus examples of bioagents which can be identified by the methods of the present invention.
  • Examples of (-)-strand RNA viral genera include arenaviruses, bunyaviruses, and mononegavirales.
  • Species that are members of the arenavirus genus include, but are not limited to, are sabia virus, lassa fever virus, Machupo Virus, Argentine hemorrhagic fever virus, and flexal virus.
  • Species that are members of the bunyavirus genus include, but are not limited, to hantavirus, nairovirus, phlebovirus, hantaan virus, Congo-Crimean hemorrhagic fever, and rift valley fever.
  • Species that are members of the monoegavirales genus include, but are not limited to, f ⁇ lovirus, paramyxovirus, ebola virus, Marburg, and equine morbillivirus.
  • Examples of (+)-strand RNA viral genera include, but are not limited to, picornaviruses, astrovirases, calciviruses, nidovirales, flaviviruses, and togaviruses.
  • Species of the picornavirus genus include, but are not limited to, coxsackievirus, echovirus, human coxsackievirus A, human echovirus, human enterovirus, human poliovirus, hepatitis A virus, human parechovirus, and human rhino virus.
  • a species of the astro virus genus includes but is not limited to, human astrovirus.
  • Species of the calcivirus genus include, but are not limited to, chiva virus, human calcivirus, and norwalk virus.
  • Species of the nidovirales genus include, but are not limited to coronavirus and torovirus.
  • Species of the flavivirus genus include, but are not limited to, Alfuy virus, Alkhurma virus, aba virus, Aroa virus, Bagaza virus, Banzi virus, Batu cave virus, Bouboui virus, Bukalasa bat virus, Bussliquara virus, Cacipacore virus, Carey island virus, Cowbone ridge virus, Dakar bat virus, Deer tick virus, Dengue virus type 1, Dengue virus type 2, Dengue virus type 3, Dengue virus type 4, Edge hill virus, Entebbe bat virus, Flavivirus sp., Gadgets gully virus, Hepatitis C virus, Iguape virus, Ilheus virus, Israel turkey meningoencephalitis virus, Japanese encephalities virus, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokobera virus, Koutango virus.
  • Kunjin virus Kyasanur forest disease virus, Langata virus, Louping III virus, Maeban virus, Modoc virus, Montana myotic leukoencephalitis virus, Murray Valley encephalitis virus, Naranjal virus, Negishi virus, Ntaya virus, Omsk hemorrhagic fever virus, Phnom-Penh bat virus, Potiskum virus, Powassan virus, Rio bravo virus, Rocio virus, Royal farm virus, Russian spring-summer encephalitis virus, Saboya virus, Saint Louis encephalitis virus, Sal vieja virus, San perlita virus, Saumarez reef virus, Sepik virus, Sitiawan virus, Sokuluk virus, Spondweni virus, Stratford virus, Tembusu virus, Tick-borne encephalitis virus, Tyulenly virus, Kenya 5 virus, Usutu virus, West Nile virus, and Yellow fever virus.
  • Species of the togavirus genus include, but are not limited to, Chikugunya virus, Eastern equine encephalitis virus, Mayaro virus, O'nyong-nyong virus, Ross river virus, Venezuelan equine encephalitis virus, Rubella virus, and hepatitis E virus.
  • the hepatitis C virus has a 5 '-untranslated region of 340 nucleotides, an open reading frame encoding 9 proteins having 3010 amino acids and a 3 '-untranslated region of 240 nucleotides.
  • the 5'-UTR and 3'- UTR are 99% conserved in hepatitis C viruses.
  • Species of retroviruses include, but are not limited to, human immunodeficiency virus and hepatitis B virus.
  • the target gene is an RNA-dependent RNA polymerase or a helicase encoded by (+)-strand RNA viruses, or RNA polymerase from a (-)- strand RNA virus.
  • (+)-strand RNA viruses are double stranded RNA and replicate by RNA- directed RNA synthesis using RNA-dependent RNA polymerase and the positive strand as a template.
  • Helicase unwinds the RNA duplex to allow replication of the single stranded RNA.
  • These viruses include viruses from the genera picornaviridae, togaviridae, flaviviradae, arenaviridae, cononaviridae (e.g., human respiratory virus) and Hepatitis A virus.
  • the genes encoding these proteins comprise variable and highly conserved regions which flank the variable regions. The genes can be used to identify the species of the virus and if necessary the strain of the viral species.
  • RNA viruses are identified by first obtaining RNA from an RNA virus, obtaining corresponding DNA from the RNA via reverse transcription, amplifying the DNA to obtain one or more amplification products using one or more pairs of oligonucleotide primers that bind to conserved regions of the RNA viral genome, which flank a variable region of the genome, determining the molecular mass or base composition of the one or more amplification products and comparing the molecular masses or base compositions with calculated or experimentally determined molecular masses or base compositions of known RNA viruses wherein at least one match identifies the RNA virus.
  • the RNA virus is a coronavirus.
  • the coronavirus includes but is not limited to, a member of the following group of coronaviruses: avian infectious bronchitis, bovine coronavirus, canine coronavirus, feline infectious peritonitis virus, human coronavirus 229E, human coronavirus OC43, murine hepatitis virus, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, rat sialodacryoadenitis coronavirus, SARS coronavirus, transmissible gastroenteritis virus and turkey coronavirus.
  • the intelligent primers produce bioagent identifying amplicons within stable and highly conserved regions of coronaviral genomes.
  • the advantage to characterization of an amplicon in a highly conserved region is that there is a low probability that the region will evolve past the point of primer recognition, in which case, the amplification step would fail.
  • Such a primer set is thus useful as a broad range survey-type primer.
  • an example of a highly conserved region of coronaviruses is the gene encoding RNA-dependent RNA polymerase (RdRp).
  • the intelligent primers produce bioagent identifying amplicons in a region which evolves more quickly than the stable region described above.
  • characterization bioagent identifying amplicon corresponding to an evolving genomic region is that it is useful for distinguishing emerging strain variants.
  • an example of an evolving genomic region of coronaviruses is the gene encoding nspl l.
  • the present invention also has significant advantages as a platform for identification of diseases caused by emerging coronaviruses.
  • the present invention eliminates the need for prior knowledge of sequence to generate hybridization probes.
  • the present invention provides a means of determining the etiology of a coronavirus infection when the process of identification of coronaviruses is carried out in a clinical setting and, even when the coronavirus is a new species never observed before (as used herein, the term "etiology" refers to the causes or origins, of diseases or abnormal physiological conditions).
  • Another embodiment of the present invention also provides a means of tracking the spread of any species or strain of coronavirus when a plurality of samples obtained from different locations are analyzed by the methods described above in an epidemiological setting.
  • a plurality of samples from a plurality of different locations are analyzed with primers which produce bioagent identifying amplicons, a subset of which contain a specific coronavirus.
  • the corresponding locations of the members of the coronavirus-containing subset indicate the spread of the specific coronavirus to the corresponding locations.
  • the present invention also provides kits for carrying out the methods described herein.
  • the kit may comprise a sufficient quantity of one or more primer pairs to perform an amplification reaction on a target polynucleotide from a bioagent to form a bioagent identifying amplicon.
  • the kit may comprise from one to fifty primer pairs, from one to twenty primer pairs, from one to ten primer pairs, or from two to five primer pairs.
  • the kit may comprise one or more primer pairs recited in Table 2.
  • the kit may comprise broad range survey primers, division wide primers, or drill-down primers, or any combination thereof.
  • a kit may be designed so as to comprise particular primer pairs for identification of a particular bioagent.
  • a broad range survey primer kit may be used initially to identify an unknown bioagent as a coronavirus. Another kit may be used to distinguish any coronavirus from any other coronavirus. In some embodiments, any of these kits may be combined to comprise a combination of broad range survey primers and division-wide primers so as to be able to identify the species of an unknown bioagent.
  • the kit may also comprise a sufficient quantity of reverse transcriptase, a DNA polymerase, suitable nucleoside triphosphates (including any of those described above), a DNA ligase, and/or reaction buffer, or any combination thereof, for the amplification processes described above.
  • a kit may further include instructions pertinent for the particular embodiment of the kit, such instructions describing the primer pairs and amplification conditions for operation of the method.
  • a kit may also comprise amplification reaction containers such as microcentrifuge tubes and the like.
  • a kit may also comprise reagents for isolating bioagent nucleic acid, including, for example, detergent.
  • a kit may also comprise a table of measured or calculated molecular masses and/or base compositions of bioagents using the primer pairs of the kit.
  • the present invention is also directed to methods of characterizing a double etiology of a subject presenting at least one symptom of SARS comprising: contacting nucleic acid from a sample from the subject with a first pair of oligonucleotide primers which hybridize to conserved sequences of a coronavirus, wherein said conserved sequences of a coronavirus flank a variable nucleic acid sequence; contacting nucleic acid from the sample with a second pair of oligonucleotide primers which hybridize to conserved sequences of a putative secondary bioagent(s), wherein the sequences of putative secondary bioagents flank a variable sequence; amplifying the variable nucleic acid sequences between the first pair of primers and the second pair of primers to produce a coronavirus amplification product and a secondary bioagent amplification product; determining the base composition signature of each of the amplification products; using the base composition signatures of each of the amplification products to identify the combination of a
  • the secondary bioagent correlates with increased severity of the at least one symptom of SARS. In some embodiments, the secondary bioagent correlates with increased incidence of mortality of subjects presenting the at least one symptom of SARS. In some embodiments, the at least one symptom of SARS is high fever (>38°C), dry cough, shortness of breath, headache, muscular stiffness, loss of appetite, malaise, confusion, rash, or diarrhea, or any combination thereof. In some embodiments, the double etiology comprises a synergistic viral infection of a SARS-linked coronavirus and a secondary virus.
  • the secondary virus is adenovirus, parainfluenza virus, respiratory syncytial virus, measles virus, chicken pox virus, or influenza virus, or any combination thereof.
  • the double etiology comprises a synergistic viral/bacterial infection of a SARS-linked coronavirus and a secondary bacterial agent.
  • the secondary bacterial agent is Streptococcus pneumoniae, Mycoplasma pneumoniae, or Chlamydia trachomatis, or any combination thereof.
  • the contacting steps are performed in parallel. In some embodiments, the contacting steps are performed simultaneously.
  • the present invention is also directed to methods of identifying the etiology of a subject presenting at least one symptom of SARS comprising: employing the method described above to rule out the presence of a SARS-linked coronavirus in a sample, wherein lack of amplification of a SARS-linked coronavirus by the first pair of primers indicates absence of a SARS-linked coronavirus, and wherein the base composition signature of the amplification product of the second pair of primers identifies the secondary bioagent, thereby indicating the etiology of the at least one symptom of SARS.
  • the secondary bioagent is the cause of an acute respiratory infection.
  • the secondary bioagent is a bacterial agent such as, for example, Streptococcus pneumoniae, Mycoplasma pneumoniae or Chlalmydia trachomatis.
  • the secondary bioagent is a viral agent such as, for example, adenoviruses, parainfluenza, respiratory syncytial virus, measles virus, chicken pox virus, or influenza virus.
  • Example 1 Coronavirus Samples, Nucleic Acid Isolation and Amplification
  • HRT-18 and MRC5 cell lines were inoculated with HCoV-OC43 and HcoV-229E (University of Colorado and Naval Health Research Center, San Diego, CA), HcoV-229E.
  • SARS RNA was obtained the CDC (Atlanta, GA) as a 1 mL extract of SARS coronavirus in TRIzol extraction buffer.
  • the SARS CoV-Tor2 strain was obtained from the University of Manitoba as a cell culture supernatant from infected Vero-E6 cells.
  • RNA was isolated from 250 ⁇ L of coronavirus infected cells or culture supernatant using Trizol or Trizol LS respectively (Invitrogen Inc., Carlsbad, CA) according to the manufacturer's protocol. 5 ⁇ g of sheared poly A DNA was added for the precipitation of the RNA. The pelleted nucleic acids were washed in 70% ethanol and resuspended in 100 ⁇ L DEPC-treated water containing 20 units of Superase » InTM (Ambion, Austin, TX). The resuspended RNA was purified using the Qiagen RNAeasy mini kit according to the manufacturer's protocol. The RNA was eluted from the RNAeasyTM columns in 30 ⁇ L of DEPC- treated water and was stored at -7O 0 C.
  • PCR reaction buffer consisted of 4 units of Amplitaq Gold, Ix buffer II (Applied Biosystems, Foster City, CA), 2.0 mM MgCl 2 , 0.4 M betaine, 800 ⁇ M dNTP mix, and 250 nM propyne containing PCR primers.
  • the following PCR conditions were used to amplify coronavirus sequences: 95oC for 10 min followed by 50 cycles of 95 0 C for 30 sec, 5O 0 C for 30 sec, and 72 0 C for 30 sec.
  • the mass spectrometer is based on a Bruker Daltonics (Billerica, MA) Apex II 7Oe electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer (ESI-
  • Ions were formed via electrospray ionization in a modified Analytica (Branford, CT) source employing an off axis, grounded electrospray probe positioned ca. 1.5 cm from the metalized terminus of a glass desolvation capillary. The atmospheric pressure end of the glass capillary is biased at 6000 V relative to the ESI needle during data acquisition. A counter-current flow of dry N 2 /O 2 was employed to assist in the desolvation process. Ions were accumulated in
  • an external ion reservoir comprised of an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode, prior to injection into the trapped ion cell where they were mass analyzed.
  • Spectral acquisition was performed in the continuous duty cycle mode whereby ions were accumulated in the hexapole ion reservoir simultaneously with ion detection in the trapped ion cell.
  • the ions were subjected to a 1.6 ms chirp excitation corresponding to 8000 - 500 m/z.
  • Data was acquired over an m/z range of 500 - 5000 (IM data points over a 225K Hz bandwidth).
  • Two target regions were selected in coronavirus orf-lb, one in the RNA-dependent RNA polymerase (RdRp) and the other in Nspl l ( Figure 7). Locations of primers within these regions were optimized for sensitivity and broad-range priming potential simultaneously by performing limiting dilutions of multiple, diverse coronaviruses.
  • the primer pair names shown in Table 2 refer to the forward and reverse primer for a given region.
  • Each primer was designed to include a thymidine (T) nucleotide on the 5' end to minimize addition of non-templated adenosine (A) during PCR.
  • Table 2 represents the collection of intelligent primers (SEQ ID NOs:5-ll) designed to identify coronaviruses using the method of the present invention.
  • the forward or reverse primer name indicates the gene region of coronavirus genome to which the primer hybridizes relative to a reference sequence, in this case, the human coronavirus 229E sequence.
  • the primers represented by SEQ ID NOs: 5 and 6 were designed to yield an amplicon originating from a coronavirus nspl l gene with reference to GenBank Accession No: NC_002645 (incorporated herein as SEQ ID NO:30).
  • SEQ ID NOs:7-l 1 The primers represented by SEQ ID NOs:7-l 1 were designed to yield an amplicon originating from a coronavirus RNA-dependent RNA polymerase gene with reference to GenBank Accession No: AF304460 (incorporated herein as SEQ ID NO: 31).
  • SEQ ID NO: 31 GenBank Accession No: AF304460 (incorporated herein as SEQ ID NO: 31).
  • @ 5-propynyluracil (which is a chemically modified version of T);
  • & 5- propynylcytosine (which is a chemically modified version of C).
  • RNA structure search algorithm T. Macke et al., Nuc. Acids Res. 29, 4724 (2001) was modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (J. SantaLucia, Proc. Natl. Acad. Sci. U.S.A 95, 1460 (1998)).
  • ePCR was used first to check primer specificity and the selected primer pairs were searched against GenBank nucleotide sequence database for matches to the primer sequences.
  • coronavirus primers should prime all known coronaviruses in GenBank, but should not prime bacterial, viral, or human DNA sequences. For each match, A, G, C, and T base counts of the predicted amplicon sequence were calculated and a database of coronavirus bioagent identifying amplicons was created (Table 3).
  • Table 3 Shown in Table 3 are molecular masses and base compositions of both strands of bioagent identifying amplicons for a series of different coronaviruses obtained using primer sets CV_NC002645_18190_18215P_F (nspll primer set SEQ ID NOs: 5 and 6) and VPOL_AF304460_1737_1755P_F (RdRp primer set SEQ ID NOs: 9 and 10).
  • Table 3 Database of Molecular Masses and Base Compositions for Coronavirus Bioagent
  • Example 5 Characterization of Bioagent Identifying Amplicons for Coronaviruses
  • two PCR primer target regions in orf-lb, one in the RNA-dependent RNA polymerase (RdRp) and the other in Nspl l were identified based on the analyses described in Examples 3 and 4. Locations of primers within these regions were optimized both for sensitivity and broad-range priming potential simultaneously by performing limiting dilutions of multiple, diverse coronaviruses. Analysis of the final primer pairs by ePCR of GenBank nucleotide database sequences showed that these primers would be expected to amplify all the known coronaviruses but no other viruses, bacteria, or human DNA.
  • FIG. 8 is a schematic representation of electrospray ionization, strand separation, and the actual charge state distributions of the separated sense and antisense strands, and determination of molecular mass and base composition of the PCR products from the RdRp primer pair for the SARS coronavirus.
  • FIG 10 Shown in Figure 10 is a spatial representation of the base compositions of five different coronaviruses for RdRp and nspl l bioagent identifying amplicons.
  • the G content of each base composition is represented by the tilt angle of the cone which indicates experimentally determined base composition.
  • Calculated base compositions are indicated by the labeled spheres.
  • Figure 10 indicates that the experimentally determined base compositions generally match the calculated base compositions.
  • One exception is the canine coronavirus analysis which indicated that a single T to C substitution (single nucleotide polymorphism) exists in the amplicon.
  • bioagent identifying amplicon does not require prior knowledge of sequence. This feature is exemplified for the bioagent identifying amplicons obtained with the nspl l primer set. No sequence was available in the nspl l region for three of the five viral species (FIPV, CcoV and HcoV OC43). Nevertheless, base compositions of the three bioagent identifying amplicons were determined which were well within the expected bounds of base compositions of coronavirus nspll bioagent identifying amplicons. Thus, had the identity of these three coronaviruses been unknown and if they had been tested with the same primer sets, they would have been identified as newly discovered coronaviruses.
  • SARS coronavirus was handled in a P3 facility by investigators wearing forced air respirators. Equipment and supplies were decontaminated with 10% hypochlorite bleach solution for a minimum of 30 minutes or by immersion in 10% formalin for a minimum of 12 h and virus was handled in strict accordance with specific Scripps Research Institute policy.
  • SARS CoV was cultured on sub confluent Vero-E6 cells at 37°C, 5% CO 2 in complete DMEM with final concentrations of 10% fetal bovine serum (Hyclone), 292 ⁇ g/mL L-Glutamine, 100 U/mL penicillin G sodium, 100 ⁇ g/mL streptomycin sulfate (Invitrogen), and 10 mM HEPES (Invitrogen).
  • Virus-containing medium was collected during the peak of viral cytopathic effects, 48 h after inoculation with approximately 10 PFU/cell of SARS CoV from the second passage of stock virus. Infectious virus was titered by plaque assay. Monolayers of Vero-E6 cells were prepared at 70-80% confluence in tissue culture plates. Serial tenfold dilutions of virus were prepared in complete DMEM. Medium was aspirated from cells, replaced by 200 ⁇ L of inoculum, and cells were incubated at 37°C, 5% CO 2 for 1 hour. Cells were overlaid with 2-3 mL/well of 0.7% agarose, Ix DMEM overlay containing 2% fetal bovine serum.
  • the SARS coronavirus stock solution was analyzed using internally calibrated PCR.
  • Synthetic DNA templates with nucleic acid sequence identical in all respects to each PCR target region from SARS CoV with the exception of 5 base deletions internal to each amplicon were cloned into a pCR-Blunt vector (Invitrogen, Carlsbad, CA).
  • the calibrant plasmid was quantitated using OD 260 measurements, serially diluted (10-fold dilutions), and mixed with a fixed amount of post- reverse transcriptase cDNA preparation of the virus stock and analyzed by competitive PCR and electrospray mass spectrometry.
  • Each PCR reaction produced two sets of amplicons, one corresponding to the calibrant DNA and the other to the SARS cDNA. Since the primer targets on the synthetic DNA calibrant and the viral cDNA were almost identical, it was assumed that similar PCR efficiencies exist for amplification of the two products. Analysis of the ratios of peak heights of the resultant mass spectra of the synthetic DNA and viral cDNA for each dilution of the calibrant were used to determine the amounts of nucleic acid copies (as measured by calibrant molecules) present per PFU, post reverse transcriptase.
  • a PFU plate forming unit
  • a PFU is defined as a quantitative measure of the number of infectious virus particles in a given sample, since each infectious virus particle can give rise to a single clear plaque on infection of a continuous "lawn" of bacteria or a continuous sheet of cultured cells. Since all of the extracted RNA was used in the reverse transcriptase step to produce the viral cDNA, the approximate amount of nucleic acids associated with infectious virus particles in the original viral preparation was estimated. To determine the relationship between PFU and copies of nucleic acid, the virus stock was analyzed using internally calibrated PCR.
  • Synthetic DNA templates with nucleic acid sequence identical in all respects to each PCR target region from SARS CoV with the exception of 5 base deletions internal to each amplicon were cloned into a pCR-Blunt vector (Invitrogen, Carlsbad, CA).
  • the calibrant plasmid was quantitated using OD260 measurements, serially diluted (10-fold dilutions), and mixed with a fixed amount of post-reverse transcriptase cDNA preparation of the virus stock and analyzed by competitive PCR and electrospray mass spectrometry.
  • Each PCR reaction produced two sets of amplicons, one corresponding to the calibrant DNA and the other to the SARS cDNA.

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Abstract

L'invention concerne un procédé d'identification et de quantification rapides de bactéries par amplification de segment d'acide nucléique bactérien, puis analyse en spectrométrie de masse. On décrit des compositions permettant de caractériser les masses moléculaires et les compositions de base d'acides nucléiques bactériens pour l'identification rapide de bactéries.
PCT/US2004/012671 2003-04-26 2004-04-23 Procedes d'identification de coronavirus Ceased WO2004111187A2 (fr)

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JP2007523629A (ja) 2007-08-23
SG161740A1 (en) 2010-06-29
US8057993B2 (en) 2011-11-15
WO2004111187A3 (fr) 2007-11-29
EP1623013A2 (fr) 2006-02-08
AU2004248107A1 (en) 2004-12-23
CA2521508A1 (fr) 2004-12-23
SG178624A1 (en) 2012-03-29
EP1623013A4 (fr) 2008-08-27

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